1. Field of the Invention
The present invention relates to flow cytometers and flow cytometry for analyzing and sorting individual particles. More particularly, the present invention relates to sample introduction apparatus for a flow cytometer, the sample introduction apparatus including a syringe directly coupled to the flow cytometer.
2. Background Art
Flow cytometers have been in clinical and research use for many years. Basically, the systems act to position small amounts of a substance within a sheath fluid. This sheath fluid may either form droplets or may exist in a stream for optical analysis. Through hydrodynamic focusing and laminar flow, the sample is forced into a single file of individual cells and the like and is surrounded by a sheath fluid. In many applications, the sheath fluid together with its entrained substance exits a nozzle in a jet and either free falls or is channeled in an optically transparent pathway for analysis. Analysis and possible sorting of the particles as they traverse the optically transparent pathway have been employed in the determination of the variety of characteristics of individual particles. This analysis is most useful in analyzing or determining characteristics of biological cells for the collection of information which would be useful in areas of research, hematology, immunology and the like. The researcher, for example, may be interested in determining specific characteristics of the individual cells so that such cells may be classified, identified, quantified and perhaps sorted for further investigations or analysis.
FIG. 1 is a schematic illustration of the operation of a typical flow cytometer. The flow cytometer 1 shown in FIG. 1 includes a nozzle system 42 that acts to introduce a flow of a substance within a sheath fluid. To accomplish this, the nozzle system 42 includes nozzle container 2 which establishes nozzle volume 3. The nozzle volume 3 has sheath fluid port 24 within it so that a sheath fluid may be introduced to it from a sheath reservoir 5 via a sheath fluid line 4. In addition, a substance introduction port 9 is included so that a substance may be introduced from substance reservoir 8 into the sheath fluid. The sheath fluid and its entrained substance are then hydrodynamically focused so that single cells and the like may be emitted from a nozzle tip 6 into free fall area 7 or an analysis area. By allowing the sheath fluid to exit from the nozzle volume 3, a jet 12 is created. This jet occurs within free fall area 7 or an analysis area where it may be analyzed or further processed.
In a sorting flow cytometer, as shown in FIG. 1, a vibration in jet 12 may be initiated by an oscillator 10. Oscillator 10 acts to initiate variations within jet 12 so that its oscillations may act to form droplets 13 from the sheath fluid at droplet separation point 69 as those skilled in the art readily understand. Each of the droplets 13 may be differentially analyzed in an analysis area to assess whether they contain a particular item or substance such as a cell or a cell fragment. For a sorting application, sorting equipment may be included to differentially charge each droplet which contains a cell or particle of interest and thus deflect them electrostatically. Typically, the sorting equipment comprises an electrical charging apparatus 16 including an electrode penetrating volume 3 and one or more electrostatic deflection plates 26. If oscillator 10 is not present or not operational, then the flow cytometer produces a continuous jet flow. In this case, the jet may simply be analyzed in an analysis area and collected in some receptacle for eventual disposal.
Importantly, most flow cytometers act to sense a specific property of the substance being analyzed. As shown in FIG. 1, this occurs through the use of some type of substance property sensor 62. Substance property sensor 62 may be positioned so that it senses light emissions occurring from the substance within jet 12. The signals then sensed may be utilized by analysis equipment 15 in a variety of different ways depending upon the particular substance or application involved. The light emissions from the substance may occur at one wavelength or frequency or may occur throughout a substance wavelength band. In order to analyze the substance, it is frequently common to cause the substance to fluoresce and then analyze its emissions. This may be created naturally or by stimulating the substance after it exits from nozzle container 2 through some type of substance stimulation source 65. Substance stimulation source 65 should be directed towards the substance possibly at location of jet 12. As those skilled in the art would readily understand, substance stimulation source 65 may emit electromagnetic radiation and thus serve as an electromagnetic source such as a laser which causes the substance to fluoresce. This fluorescence may occur within a fluorescence emission band.
A known problem in the use of conventional flow cytometers arises from cross contamination between successive sample substances and contamination from non-sterile instrument parts. For example, FIG. 2 illustrates a portion of the fluidics system of a conventional flow cytometer. As illustrated in FIG. 2, the nozzle container 2 receives a sheath fluid received from sheath fluid lines 4 via sheath fluid ports 24 and receives a substance to be analyzed from substance introduction port 9 via a substance introduction port 9. Sheath fluid exits the nozzle container 2 through the nozzle tip 6. The reservoir 8 of the substance to be analyzed is contained within a pressurized container 20. Gas pressure is introduced into pressurized container 20 from gas line 18 and the pressure is controlled or shut off by gas pressure valve 19. The pressure in the container 20 forces the substance of the reservoir 8 to be forced into the substance introduction port 9 and the flow of the substance within the substance introduction port 9 is controlled or shut off by valve 17. Alternatively, instead of using a pressurized vessel 20, the sample substance may be caused to flow within the substance introduction port by means of a mechanical pump, such as a syringe pump.
Within the conventional flow cytometer diagrammatically illustrated in FIG. 2, to remove the reservoir comprising a first substance so that a second substance may be analyzed in its place, the valve 17 and the valve 19 are shut off so as to isolate the container 20. The container 20 is then replaced with another container having a reservoir of the second substance. Also, the substance introduction port 9 and the valve 17 must be either replaced or else thoroughly cleaned and sterilized. The non replaceable elements of the nozzle container 2 must be thoroughly flushed and sterilized. When replacing the various parts, such as the container 20 or the sample introduction port, care must also be exercised to ensure that the replacement parts are clean and sterile.
The above-described precautions that must be undertaken to prevent the contamination of an analyzed substance or to prevent cross contamination between substances can decrease the overall usage efficiency of a conventional flow cytometer. The exercise of these contamination precautions can also delay the start of and increase the costs of analyses obtained from the conventional flow cytometer. Occasionally, within clinical or hospital settings, important or even critical patient treatment decisions depend upon the rapid completion of analyses of biological samples, such as blood or other bodily fluids. Thus, inefficiencies in analytical capabilities can have detrimental health consequences for patients. Furthermore, these biological specimens are often withdrawn from a patient using a syringe and then subsequently transferred to holding vessels, such as the pressurized container 20 (FIG. 2) of a conventional flow cytometer. Each such transfer creates new opportunities for contamination or possible spoilage of the sample. Both time and contamination problems could be reduced if the specimen is transferred to the analytical instrument, such as a flow cytometer, in the same syringe used to obtain the specimen.
As a result of the above considerations, there is a need in the art for a simplified sample introduction system for a flow cytometer that eliminates many of the sample transfer lines, vessels and valves, that can reduce the preparation time required to initiate an analysis with the flow cytometer and that allows a greater number of samples to be analyzed in a given time with minimal contamination concerns. The present invention addresses such a need.